Allosteric gate modulation confers K ⁺ coupling in glutamate transporters

Abstract

Excitatory amino acid transporters (EAATs) mediate glial and neuronal glutamate uptake to terminate synaptic transmission and to ensure low resting glutamate concentrations. Effective glutamate uptake is achieved by cotransport with 3 Na+ and 1 H+, in exchange with 1 K+. The underlying principles of this complex transport stoichiometry remain poorly understood. We use molecular dynamics simulations and electrophysiological experiments to elucidate how mammalian EAATs harness K+ gradients, unlike their K+‐independent prokaryotic homologues. Glutamate transport is achieved via elevator‐like translocation of the transport domain. In EAATs, glutamate‐free re‐translocation is prevented by an external gate remaining open until K+ binding closes and locks the gate. Prokaryotic GltPh contains the same K+‐binding site, but the gate can close without K+. Our study provides a comprehensive description of K+‐dependent glutamate transport and reveals a hitherto unknown allosteric coupling mechanism that permits adaptions of the transport stoichiometry without affecting ion or substrate binding.

Na + -dependent gate dynamics and electrostatic attraction ensure substrate coupling in glutamate transporters

“…By mutating two of these residues (R397 and M311) to alanine, we effectively decoupled Na + binding from aspartate association, revealing their central role in controlling HP2 conformation. HP2 dynamics differ in Glt Ph and EAAT1: Whereas HP2 is mostly open in apo EAAT1, it can close in apo Glt Ph , permitting retranslocation and the K + -independent transport of Glt Ph (19). The distinct HP2 dynamics of Glt Ph and EAAT1 also have important implications for Na + binding.…”

“…The elevator mechanism ensures stoichiometrically coupled transport by permitting translocation of the transport domain only with certain ion and substrate occupations, i.e., in the fully bound configuration (including three Na + ions and the substrate), in the apo (in Glt X only), or in the K + -bound configuration (in EAATs). During glutamate uptake, inward translocation is made possible via induced-fit substrate binding and HP2 closure (20,21), while K + binding stabilizes HP2 in its closed conformation to enable K + -bound retranslocation of the EAATs (19). To make elevator transport effective, unproductive back-and-forth movements of the transport domain need to be prevented.…”

“…Hairpin loop 2 (HP2) serves as a gate controlling access to the substrate-binding pocket in both the outward-and inward-facing states (13)(14)(15)(16), and translocation of the transport domain is only possible with closed HP2 (14,17). HP2-gating dynamics are allosterically regulated by ion and substrate binding (17)(18)(19)(20)(21). HP2 is tightly closed in fully bound or apo transporters (16,17,22,23), whereas addition of the competitive transport blocker DL-threo-β-Benzyloxyaspartic acid (TBOA) resulted in crystallization of HP2 in the open state (16,24).…”

“…The elevator alternating-access mechanism accounts for various functional properties of the EAATs and related homologs and permits combining high substrate selectivity with complex transport stoichiometries (25,26). The separation between ion and substrate binding and translocation dynamics allows for the evolutionary adjustment of ion-coupling mechanisms without changing the binding sites or the translocation mechanism, resulting in a variety of transport stoichiometries in the SLC1 familyranging from purely Na + -or H + -coupled transporters in prokaryotes to Na + -, K + -, and H + -coupled eukaryotic glutamate transporters (EAATs) and Na + -dependent neutral amino acid exchangers (ASCTs) (19). However, certain features of elevator transport might impair effective transport at fixed stoichiometry, and how these limitations are overcome remains unclear.…”

An amino-terminal point mutation increases EAAT2 anion currents without affecting glutamate transport rates

“…This so-called elevator mechanism was first described for the prokaryotic glutamate transporter GltPh (14,15) and later shown to also apply to an increasing number of other secondary active transporters (16)(17)(18). For EAAT/GltPh, it ensures strict stoichiometric coupling of glutamate to Na + , K + and H + transport by permitting translocation only for certain ligation states of the transporter (19)(20)(21)(22). Elevator-like transport is also the basis of the anion channel function of this class of glutamate transporters.…”